Cell Discovery

Pseudomonas aeruginosa is an opportunistic human pathogen capable of infecting a wide range of tissues and organs. Its persistence during chronic infection is strongly associated with biofilm formation, which depends on extracellular polysaccharides such as alginate. The HtrA-like periplasmic serine protease MucD is a key regulator of bacterial virulence, stress response, and alginate production, yet its molecular mechanism has remained largely unclear. Here, we discovered the alginate acetylation and export proteins AlgX and AlgK as MucD substrates, and characterized their degradation by mass spectrometry and bioinformatic analysis. We further determined the cryo-EM structure of MucD bound to an AlgK-derived substrate peptide, offering atomic insights into MucD oligomerization assembly, substrate recognition, and specificity. Together with structure-guided mutagenesis and biochemical assays, our results revealed that MucD proteolytic activity is governed by an equilibrium between a resting 12-mer and an active trimer. Crucially, we demonstrate that MucD represses alginate biosynthesis post-translationally, in addition to its previously implicated role in transcriptional regulation. These findings define a distinct activation mechanism and regulatory function for MucD and provide new insight into bacterial HtrA-like serine proteases.

Deep learning-based protein structure prediction methods that leverage evolutionary information from multiple sequence alignments (MSAs), exemplified by AlphaFold2, have achieved remarkable accuracy. However, existing methods still struggle to predict challenging proteins, particularly those with novel folds or limited evolutionary information, and to recover alternative conformational states. Here we show that structure prediction models trained under different MSA-depth distributions corresponding to different levels of evolutionary information exhibit complementary generalization behaviors, and that a model trained on a mixture of these distributions can combine their complementary generalization strengths. Building on this insight, we developed ProtMonomer, a deep learning framework trained on MSA-depth distributions representing a broad range of evolutionary information levels to improve structure prediction. Across benchmarks comprising CASP15 targets, non-redundant experimentally determined structures, orphan proteins, and short peptides, ProtMonomer performed comparably to or better than leading methods, including AlphaFold2 and AlphaFold3, with particularly strong performance on challenging targets. For fold-switching proteins, ProtMonomer also recovered alternative conformational states more accurately than AlphaFold2 and AlphaFold3 across diverse homologous sequence sampling strategies. In addition to improving predictive accuracy, ProtMonomer substantially reduced inference cost through an efficient architecture, enabling high-throughput applications. Together, these findings provide insights into the generalization of evolution-informed structure prediction models and support ProtMonomer as an accurate and efficient framework for protein structure prediction.

Klebsiella pneumoniae (Kp) is a WHO high-priority pathogen for vaccine development, yet previous efforts failed largely because key protective immune mechanisms remain unclear. Here we show that protective immunity conferred by mucosal mRNA vaccines (but not parenteral) require neither serum IgG nor airway secretory IgA, but instead depends on a previously unrecognized lung-resident CD8IL-17 T-cells (Tc17) that rapidly recruits neutrophils/macrophages to eliminate bacteria. To therapeutically harness this paradigm, we developed INSPIRE, a machine learning-engineered exosome platform incorporating donor-screened, miRNA-bioactive backbones (miR-21-mediated airway barrier penetration and miR-155-associated dendritic-cell activation through SOCS1/Inpp5d axis) and computationally designed peptides that boosts 11.6-fold mRNA encapsulation and 3-fold dendritic-cell cross-presentation. Intranasal INSPIRE-mRNA vaccination confers near-complete protection against clinically relevant Kp strains while intramuscular counterparts fail (below ~30% survival). Leveraging pIgR-/- and IL-17-/- mice coupled with T-cell depletions, we demonstrate the protection is Tc17-dependent. This work overturns the antibody-centric dogma and redefines a non-canonical Tc17-correlate for extracellular bacterial pneumonia.

Seasonal influenza A viruses cause significant global morbidity each year. Although vaccination remains the primary preventive strategy, effectiveness is often reduced by antigenic drift. This challenge is particularly pronounced for influenza A(H3N2), which has required eight vaccine updates over the past decade. Here, we present a computational framework to engineer broadly reactive influenza A(H3N2) vaccines, using protein language models to generate novel hemagglutinin (HA) sequences and a machine learning model to predict antigenic distance from circulating strains. In a proof-of-concept study, seven HA candidates designed using sequence data from 2013-2018 were evaluated in mice against contemporary and subsequently circulating viruses. Two candidates elicited protective levels of reactive antibodies, robust H3-specific antibody-secreting cell responses, and cross-neutralization against contemporary clades and drifted 2019-2020 strains. These findings demonstrate that an integrated generation-selection strategy can enhance vaccine coverage across current and future A(H3N2) seasons and may be applicable to other influenza subtypes.

Bacterial genome-wide association studies (GWAS) offer a powerful approach to identify the genetic basis of a trait measured in a set of sequenced isolates. As the number of sequenced isolates has grown, the limiting factor for GWAS has become phenotyping enough isolates to achieve statistical power. To overcome the need for large-scale phenotyping, we developed Bayesian Adaptive Sequential Sampling GWAS (BASS-GWAS), which couples Bayesian adaptive experimental design with a sparse regression model to select maximally informative isolates for phenotypic testing. BASS-GWAS efficiently recovered causal loci for three antimicrobial resistance traits in Neisseria gonorrhoeae, requiring many fewer phenotyped isolates than random sampling. We applied BASS-GWAS to discover variants enabling gyrBD429N-dependent cross-resistance to the novel topoisomerase inhibitors zoliflodacin and gepotidacin. After phenotyping fewer than 30 isolates, we identified and then validated both parCD86N and a gyrA-parE-based pathway as enabling cross-resistance. BASS-GWAS provides a practical and statistically principled solution for efficient bacterial GWAS.

Hepatocellular carcinoma (HCC) is particularly aggressive and difficult to treat. Due to the lack of early clinical diagnosis and the unsatisfactory clinical treatment effect, it is particularly important to identify novel markers that can predict tumor behavior in HCC. biogenesis of ribosomes BRX1 (BRIX1) is abundant in various tissues of the human body. However, the regulatory mechanisms and its role in various tissues are not fully understood. Here, we analyzed the expression pattern of BRIX1 in HCC from public gene expression databases and tissue samples from clinical HCC. We confirmed that BRIX1 was upregulated in both HCC cell lines and HCC paraffin section samples. BRIX1 depletion significantly dicreased the capacity of cells to grow and migrate in vitro, and knockdown BRIX1 suppressed tumor growth in xenograft tumor model. Mechanistically, BRIX1 depletion suppressed the MAPK/ERK pathway, as reflected by reduced phosphorylated ERK (p-ERK) levels. In summary, we provide a rational clue for the further investigation of BRIX1 as an invaluable biological marker for diagnosing and predicting prognosis of patients with HCC.

Accurate, reproducible interpretation of kidney allograft biopsies is critical for diagnosis of graft injury to guide prognosis and management. The international Banff classification is a consensus diagnostic system based on semiquantitative histological lesion scoring on either extent or severity of kidney transplant biopsies. However, pathologist scoring is limited by substantial interobserver variability, constrained scalability, and the inherent nature of the scoring system itself. Here we present BanffNET, a weakly supervised, probabilistic deep learning framework that combines self-supervised feature extraction with a novel Bayesian multiple-instance learning framework to predict (continuously) the full spectrum of Banff lesion scores directly from whole-slide images (WSIs). Using lesion-specific aggregation functions tailored to localized (modeling lesion severity) and diffuse pathologies (modeling lesion extent), BanffNET generates interpretable, patch-level probability maps and calibrated slide-level scores. BanffNET's performance was assessed relative to consensus, biological correlates of rejection and clinical outcome, demonstrating superior consistency, transportability and generalization. Trained on 7,249 WSIs from three cohorts, BanffNET demonstrates consistent performance on 11,028 WSIs across five external test sets, performing on par or exceeding expert consensus across lesions. BanffNET scores align more closely than pathologist Banff scores with molecular profiles of rejection, offering a transparent, biologically grounded framework for computational pathology with relevance beyond transplantation.

A developmental increase in cerebrospinal fluid (CSF) production during development is essential for neuronal growth and ventricular expansion. A key regulator of CSF production is the specialized sensory multicilia of the choroid plexus (ChP), which mediate non-canonical Sonic hedgehog (Shh) signaling to suppress water channel and ion transporter expression, thereby limiting CSF production. ChP multicilia progressively shortens during development, attenuating Shh signaling and promoting CSF production. Here, we identify miR-34/449 miRNAs as essential regulators of ChP multiciliogenesis. Whereas mutations in canonical ciliogenesis genes elevate CSF production and contribute to hydrocephaly, deletion of miR-34/449 reduces CSF volume and causes microcephaly. Loss of miR-34/449 miRNAs causes excessive basal body amplification, defective basal body docking, and failure of developmental multiciliary shortening. Consequently, miR-34/449-deficient ChP cilia remain abnormally long and fail to attenuate Shh signaling, resulting in sustained repression of water channel and ion transporter expression and reduced CSF production. Mechanistically, miR-34/449 miRNAs directly target Gmnc, a master transcriptional regulator of multiciliogenesis, to restrain basal body amplification and promote basal body docking. Together, our findings identify miR-34/449 miRNAs as critical regulators of ChP multiciliogenesis and establish the developmental remodeling of ChP multicilia as a mechanism to couple Shh signaling dynamics to developmental control of CSF production.

Cyclic oligosaccharides possess industrial advantages, including molecular encapsulation capability and high physicochemical stability, owing to the absence of a reducing end. Recently, a novel cyclic tetrasaccharide, cycloisomaltotetraose (CI4), consisting of four -1,6-linked glucose units, and the enzymes responsible for its synthesis, cycloisomaltotetraose glucanotransferases (CI4Tases), were discovered. Unlike known cycloisomaltooligosaccharide glucanotransferases (CITases) that yield a wide distribution of cyclic products with a degree of polymerization (DP) of 7 or higher, CI4Tases strictly produce CI4. To elucidate the molecular mechanism underlying this strict DP4 specificity, we determined the crystal structures of CI4Tase from Agreia sp. D1110, in its ligand-free form, as well as in complex with the linear hydrolysis product isomaltotetraose (IG4) and with CI4. Structural comparisons revealed that a loop (M247 to R251) blocks the region corresponding to the -5 subsite of typical CITases, narrowing the substrate-binding pocket. This "molecular ruler" mechanism ensures that only a glycan chain of exactly four glucose units is accommodated for cyclization. Among mutants of the residue positioned at the center of bound CI4, the formation of by-products other than CI4 was significantly suppressed in F245L, F245A, and F245W. While the cyclization activity of all F245 mutants decreased, the CI4 hydrolysis activity of these three mutants was also significantly reduced, resulting in an increased specificity for cyclic sugar production. These findings elucidate the strict size-control mechanism of CI4Tase and provide a structural foundation for engineering cycloisomaltooligosaccharide-producing enzymes with optimized transglycosylation efficiency and specificity for industrial applications.

Solid tumors remain refractory to conventional treatments, yet cell surface proteins, by virtue of their extracellular accessibility and critical roles in tumor signaling, represent an attractive class of targets for precision-targeted therapy. Here, we report that TMEM132A is an essential and previously unrecognized pan-cancer target. TMEM132A interacts directly with EGFR and stabilizes its expression, thereby tethering EGFR at the plasma membrane and sustaining constitutive activation of lipid synthesis. Mechanistically, the TMEM132A-EGFR axis promotes lipogenesis by facilitating SREBP nuclear translocation, which in turn upregulates ACLY and ACSS2 expression to drive acetyl-CoA production and downstream lipid biosynthesis, ultimately disrupting lipid droplet homeostasis. To therapeutically target this axis, we developed a nanobody, LFNanoT132A#3, which effectively blocks the TMEM132A-EGFR interaction, abrogates downstream signaling activation, and potently inhibits proliferation across multiple solid tumor types. Notably, LFNanoT132A also exerts robust antitumor activity against H1975 xenografts, a model resistant to first- and second- generation EGFR inhibitors, underscoring its potential to overcome conventional drug resistance. Our findings establish TMEM132A#3 as a critical node in membrane-tethered oncogenic signaling and metabolic rewiring, and position LFNanoT132A#3 as a promising therapeutic candidate for precision cancer therapy.

Hirschsprung disease (HSCR) is a severe birth defect where ganglia of the enteric nervous system (ENS) are missing from distal bowel. The aganglionic segment is also characterized by increased epithelial permeability and pro-inflammatory immune activation. These problems may sequentially lead to translocation of gut microbes into the colon wall and systemic circulation, resulting in enterocolitis and sepsis. Current HSCR treatment via surgical resection of the aganglionic segment is lifesaving but not curative, often leaving patients with persistent gastrointestinal complications including recurrent risk of enterocolitis. As alternative, we are developing a regenerative medicine strategy based on in situ stimulation of tissue-resident ENS progenitors via rectal administration of the neurotrophic factor GDNF. Here, we report that GDNF-based therapy has pleiotropic gastrointestinal effects in a mouse model of short-segment HSCR, beyond its role in ENS regeneration. Interestingly, we found that these protective effects are not restricted to the aganglionic distal colon, also positively impacting the ENS-containing proximal colon. GDNF treatment reduces bacterial translocation both locally and in peripheral organs, and this is associated with recovery of the key epithelial junction proteins CLDN3, ZO1 and DSG2. Furthermore, multiparameter flow cytometry-based analysis of 55 lymphoid and 17 myeloid cell subtypes revealed that GDNF treatment has global anti-inflammatory effects, preferentially affecting innate over adaptive immunity. Overall, these findings highlight a critical role for GDNF treatment in reestablishing proper epithelial and immune cell homeostasis, offering promising therapeutic avenues not only for HSCR but also potentially for other intestinal disorders with overlapping pathophysiology.

Post-COVID-19 syndrome (PCS) is characterized by fatigue, neurological impairment and systemic symptoms. This heterogeneity of symptoms hinders biomarker development. Here, we profiled extracellular-vesicle (EV) surface markers in plasma and CSF from 61 participants with PCS (COVIDpost), 80 recovered controls (COVIDreco), and 10 participants with non-SARS-CoV-2 post-viral syndromes. EVs were analysed by bead-based multiplex flow cytometry using tetraspanin-directed (TSPN) and phosphatidylserine-directed lactadherin (PS) detection. Amongst 37 targets covering tetraspanins and vasculature-, immunity- and stemness-associated markers, none met a 1% false-discovery-rate threshold. However, L1-regularized logistic regression under fully nested 5x5 cross-validation identified a distributed plasma EV profile, with mean out-of-fold areas under the receiver operating characteristic curve (AUCs) of 0.788 (95% CI 0.715 - 0.852) for TSPN and 0.716 (95% CI 0.636 - 0.792) for PS detection. Across the pooled COVIDpost and COVIDreco population, EV classification scores covaried with clinical group differences, but did not track clinical severity within either cohort. These PCS-EV classification scores decreased at one-year follow-up in COVIDpost participants. Our findings identify an internally cross-validated multivariable EV surface profile associated with COVIDpost versus COVIDreco status and support independent validation and exploration of EV-based biomarkers in post-viral fatigue syndromes.

IgM is the most ancient antibody isotype, playing an important role in both circulatory and mucosal immune responses across vertebrates, yet structural characterization of its polymeric forms is limited outside of mammals. Here, we report the cryo-electron microscopy structure of mallard duck secretory (S) IgM at 3.37-[A] resolution. The structure revealed a pentameric core globally similar to human SIgM, supporting the view that pentameric IgM is subject to strong evolutionary constraints. However, compared to mammalian structures, we observed species-specific differences at molecular interfaces. Surface plasmon resonance binding assays characterizing secretory component (SC)-IgM interactions supported structural observations and, when compared to IgA binding, revealed isotype-specific contributions from the avian SC N-terminal extension. Together, these findings establish a comparative structural framework for polymeric IgM across vertebrates and provide insight into how avian SIgM-specific features may support mucosal immunity in birds.

Ammonia oxidation initiates nitrification and is closely linked to microbial N2O production. Ammonia monooxygenase (AMO) catalyzes the first and rate-limiting step of nitrification and is widespread across evolutionarily distinct ammonia-oxidizing archaea (AOA) and bacteria (AOB). The ocean is the largest biome for AOA and AOB, which have distinct ecological niches and markedly different sensitivities to nitrification inhibitors. However, the lack of archaeal AMO structures and inhibitor-bound AMO complexes has hindered mechanistic understanding of the architectural, catalytic, and inhibitory divergence between these two enzyme systems. Here, we report high-resolution cryo-electron microscopy (cryo-EM) structures of marine archaeal AMO captured in active and inactivated states within its native membrane environment, together with inhibitor-bound structures of estuarine bacterial AMO. Archaeal AMO forms an unexpected cup-shaped homotrimer composed of eight subunits per protomer and exhibits substantial architectural divergence from bacterial AMO. Integrated structural, biochemical, kinetic, and computational analyses reveal distinct periplasmic architectures, copper-center organization, and hydrophobic channels between archaeal and bacterial AMOs for ammonium acquisition, catalysis and inhibitor response. These findings provide a structural and mechanistic framework for understanding how archaeal and bacterial AMOs have diverged to distinct ammonia-oxidizing strategies and inhibitor susceptibilities across environmentally important ammonia oxidizers.

To evaluate the cost-utility and 5-year budget impact of first-line olaparib plus abiraterone versus abiraterone alone for metastatic castration-resistant prostate cancer (mCRPC) in China after the eleventh round of volume-based procurement (VBP). The intention-to-treat (ITT) population was assigned primary decision-analytic weight; the prespecified BRCA1/2-mutated (BRCAm) subgroup was a supporting analysis.

tRNAs are characterized by extensive chemical modifications that influence tRNA fate. N1-methyladenosine at position 58 (m1A58) is a widespread core tRNA modification linked to physiological and pathological processes. However, how m1A58 coordinate tRNA folding and processing to ensure translational efficiency in mammalian cells remains largely unknown. Using acute dTAG-mediated degradation and CRISPR-Cas9 knockout, we identified initiator methionine tRNA (tRNAiMet) as selectively vulnerable to m1A58 loss, lacking the isodecoder buffering observed for most other tRNA isoacceptors. NMR analysis of the tRNAiMet showed that m1A58 stabilizes D/T-loop interactions, consistent with a maturation-competent conformation. In vitro processing assays further demonstrated that m1A58 promotes RNase P-mediated 5'-leader removal and RNase Z-mediated 3'-trailer cleavage, while La/SSB protects accumulated precursors. Disrupting this checkpoint impaired the assembly of the eIF2-containing 43S pre-initiation complex and global protein synthesis, which was substantially rescued by adding m1A58-modified tRNAiMet. Acute TRMT6 degradation elicited temporally coordinated gene-expression responses involving proteostasis, transport and signaling. Together, these findings establish m1A58 as a conformational checkpoint coupling human initiator-tRNA maturation to translation initiation and stress responses.

Antral CCK2R+ stem cells are regulated by gastrin, but how endocrine and neural cues integrate under chronic injury remains unclear. Here we show that inducible hypogastrinemia shifts from asymmetric renewal to symmetric expansion of CCK2R+ stem cells. With carcinogenic stress, these cells acquire a cycling, injury responsive progenitor state revealed by single-cell RNA profiling. Acute gastrin loss activates a CCK2R+ nodose DMV vagal reflex that increases acetylcholine release, NGF production, cholinergic innervation, and Chrm3 expression, driving ERK and YAP signaling in CCK2R+ stem cells. Vagotomy, Trk inhibition, or Chrm3 deletion each suppressed stem cell expansion. In H. pylori and MNU injury models, hypogastrinemia amplified inflammation, dysplasia, and CCK2R+ clone expansion, whereas gastrin suppressed these responses. Human scRNA seq and spatial profiling confirmed G cell depletion and progenitor state enrichment. These findings define an endocrine neural epithelial axis in which gastrin loss boosts vagal M3R signaling to initiate antral preneoplasia, highlighting this pathway for early interception.

We report the generation and characterization of a human induced pluripotent stem cell (iPSC) line derived from dermal fibroblasts of a patient with Skogholt disease, a rare maternally inherited neurodegenerative syndrome associated with choroid plexus dysfunction and impaired cerebrospinal fluid (CSF) homeostasis. Patient fibroblasts were reprogrammed using the non-integrating Repro-OSKGM kit. The resulting iPSC line exhibited typical pluripotent morphology, expressed canonical pluripotency markers, maintained a normal karyotype, retained the disease-associated genetic variant, was mycoplasma-free, and demonstrated trilineage differentiation potential. We also made choroid plexus (ChP) like organoids from the generated iPSCs. This patient-specific iPSC line provides a valuable resource for generating choroid plexus organoids and neurons to investigate disease mechanisms and develop therapeutic strategies.

A comprehensive recording of cell fate transitions and underlying molecular changes remains a fundamental goal in developmental biology. Here, we present DeepTrack, a lineage tracing mouse model that integrates in situ cellular barcoding with high-throughput, single-cell multi-omics to simultaneously profile clonal fates, transcriptomic states, and chromatin accessibility. Using DeepTrack, we profiled clonal behaviors during gastrulation and early organogenesis, uncovered early fate priming within epiblast clones, and revealed clonal architecture within distinct regions of the nervous system. Embryo-wide multi-omic lineage tracing at single-cell resolution revealed transcriptional and epigenetic programs underlying fate commitment in neuromesodermal progenitors (NMPs). Clonal tracing with multi-omic profiles enabled inference of fate-associated gene-regulatory networks and identified the transcription factor Cdx2 as a key regulator of mesodermal specification in NMPs. Genetic perturbation of Cdx2 in chimeric embryos impaired paraxial mesoderm differentiation. Together, DeepTrack provides a versatile framework for decoding multimodal regulation of cell fate across diverse developmental contexts.

Lineage-restricted transcriptional programs establish cell identity and can create selective dependencies in cancer. Here, we identify POU2AF2, encoding the transcriptional co-activator OCA-T1, as a critical lineage-specific dependency in a subset of diffuse large B-cell lymphoma (DLBCL). Pan-cancer dependency analyses and patient cohorts reveal elevated POU2AF2 expression in genetically aggressive DLBCL, where its depletion markedly suppresses tumor growth in vitro and in vivo. Mechanistically, POU2AF2 cooperates with the B-cell lineage-defining transcription factor POU2F2 (OCT2) to activate lymphocyte activation gene programs through direct chromatin engagement, thereby sustaining malignant transcriptional networks. We further identified a key epigenetic regulatory axis composed of the lineage-specific transcription factor TCF3 and the histone methyltransferase SET1A-COMPASS that drives POU2AF2 expression downstream of B-cell receptor signaling. Single-cell transcriptomic analysis reveals that POU2AF2 marks and sustains an innate-like B1 B-cell population in vivo, a candidate cell of origin for lymphoma. Together, these findings define a lineage-restricted POU2AF2/POU2F2 transcriptional module, controlled by a TCF3/SET1A epigenetic network, that sustains both innate-like B-cell identity and malignant fitness in DLBCL. Our study uncovers a previously unrecognized lineage-specific transcriptional dependency and highlights POU2AF2 and its associated regulatory circuitry as potential therapeutic targets in aggressive B-cell malignancies.